HER2/neu is a member of EGF family and has an important role in breast cancer. Blocking of HER2 by monoclonal antibody (Trastuzumab:Herceptin) has become a standard therapy in HER2 over-expressing breast cancer. RNA interference is a posttranscriptional gene silencing phenomenon that small ds RNA can block the expression of its corresponding gene with high specificity. Transfection of dsRNA of 19-21nt, most commonly used, is not suitable for cancer gene therapy due to its low transfection rate and short duration. Adenovirus is an ideal vector for cancer gene therapy because of its high infectivity and high expression rate. In this study, we developed an adenovirus expressing siRNA to HER2/neu. We selected 3 can... More
HER2/neu is a member of EGF family and has an important role in breast cancer. Blocking of HER2 by monoclonal antibody (Trastuzumab:Herceptin) has become a standard therapy in HER2 over-expressing breast cancer. RNA interference is a posttranscriptional gene silencing phenomenon that small ds RNA can block the expression of its corresponding gene with high specificity. Transfection of dsRNA of 19-21nt, most commonly used, is not suitable for cancer gene therapy due to its low transfection rate and short duration. Adenovirus is an ideal vector for cancer gene therapy because of its high infectivity and high expression rate. In this study, we developed an adenovirus expressing siRNA to HER2/neu. We selected 3 candidate sequences of HER2/neu for RNAi using siRNA target finder program (Genscript). After the annealing of oligonucleotides containing antisense, loop sequence and sense sequence, ds ODN was cloned into pRNAT-H1.1/shuttle (SD1216: Genscript). After digestion with Pl-SceI/I-CeuI, it was cloned into BD adeno-X viral DNA via in vitro ligation(in vitro homologous recombination). Resulting adenoviral genome containing H1 promotor-sense(19bp)-loop-antisense(19bp) were transfected into 293 cells. Generation of adenoviruses containing siHER2/neu (3034, 395 and 465) was easily confirmed by expression of Green Fluorescent Protein (GFP). Transduction with adenovirus-siHER2/neu(3034) effectively decreased the expression of HER2/neu in two breast cancer cell lines (SK-BR3 and MCF7). Down-regulation of HER2/neu by adenovirus-siHER2/neu(3034) induced the reduction of soft agar clonogenicity of breast cancer cell line (SK-BR3). Pretreatment of ad-siHER2/neu increased the sensitivity of SK-BR3 to gemcitabine, most effective anticancer drug for breast cancer. We concluded that adenovirus mediated siRNA to HER2/neu delivery to cancer cell had a potential to become a novel gene therapy method and this adenovirus mediated siRNA can be extended to other gene target for cancer treatment.
